We isolated subclones of Huh7 cells and demonstrated that they differed greatly in their ability to be transfected with hepatitis E virus (HEV) genomes or to be infected with HEV virions. We used one of the subclones to confirm that monoclonal antibodies presumed to be neutralizing antibodies based on indirect evidence did indeed neutralize HEV. We found that transfection of this subclone resulted in production of virions that could be extracted from the cells and used to infect new cells. Therefore, this system was utilized to study recombinant viral mutants. In particular, we demonstrated that ORF3 protein is not a structural component of virions: ORF3 protein was not required for infection, replication, or assembly of HEV in cultured Huh7 cells and virions produced in the absence of ORF3 protein could not be distinquished from those made in its presence. We demonstrated that the HEV replication strategy involves synthesis of a single subgenomic RNA which is a bicistronic messenger RNA that encodes both the ORF3 protein and the capsid protein. We identified heat shock cognate protein 70 as a participant in the process by which HEV enters into Huh7 cells. We developed an interferon-gamma ELISPOT assay to characterize the hepatitis E-specific cell-mediated immune response.